A major bottleneck in protein expression and purification is the costs associated with functional protein production in biomedical, biopharmaceutical and other research fields. Because proteins of interest originating from eukaryotic organisms often require specific components for post- translational events to mature into active forms, their functional production is crucially dependent on eukaryotic expression systems. However, most of these systems express eukaryotic proteins of interest only at relatively low yields and thus do not deliver sufficient amounts of active proteins to satisfy academic and industrial demands at reasonable costs. Bacterial hosts express proteins in enhanced amounts if the proteins are genetically fused to various protein tags, including ubiquitin family members (including the ubiquitin-like protein SUMO). A generally reliable fusion technology for quantitatively and qualitatively enhanced heterologous protein expression similar to fusions for bacterial expression has not yet been available for use in eukaryotic cells. LifeSensors' SUMO fusion technology, which is highly successful in bacterial hosts, is not yet applicable to eukaryotic cells, since these cells contain native SUMO proteases that cleave SUMO from its fusion partner in vivo. LifeSensors proposes to develop a SUMO fusion technology for functional protein production and purification in eukaryotic cells in a two step Phase I. First, a human SUMO3 fusion tag will be engineered that is not cleavable in eukaryotic host cells by mutagenizing the interaction domain of the SUMO variant with human deSUMOylase SENP2; next, a SUMO protease that can cleave the mutant SUMO tag in vitro to release the expressed protein will be engineered. Thus, protein expressed in enhanced yield but uncleaved in the host cell can be purified from the cells as a tagged fusion and subsequently cleaved to give pure protein. PUBLIC HEALTH RELEVANCE: A major bottleneck in protein expression and purification is the costs associated with functional protein production in biomedical, biopharmaceutical and other research fields. Increasing the quantity by several-fold would alleviate this problem considerably. This can be accomplished in bacteria by expressing the protein as a fusion, that is, a protein of interest joined to another protein (called SUMO). The fusion can be isolated from bacterial cells and then the SUMO part can be removed by an enzyme (protease) that recognizes SUMO. Many proteins need to be expressed in mammalian cells, unfortunately, because bacteria do not have the machinery to modify the expressed proteins and make them active. The SUMO method developed for bacteria does not work, however, for proteins expressed in eukaryotic cells, because, unlike bacteria, these cells contain enzymes that remove SUMO from the fusion before the fusion can be purified away from the other cellular proteins. LifeSensors, which developed the SUMO system for bacteria, proposes to engineer specially mutated versions of 1)SUMO that cannot be cut inside the cell by native enzymes, and 2) a mutated protease enzyme that can cut the mutated SUMO outside the cell when the fusion is purified. This combination will allow the same kind of SUMO-enhanced protein production as that afforded by bacterial systems. [unreadable] [unreadable] [unreadable]